title: High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA.
abstract: MicroRNAs (miRNAs) are an emerging class of small non-coding RNAs implicated in a wide variety of cellular processes. Research in this field is accelerating, and the growing number of miRNAs emphasizes the need for high-throughput and sensitive detection methods. Here we present the successful evaluation of the Megaplex reverse transcription format of the stem-loop primer-based real-time quantitative polymerase chain reaction (RT-qPCR) approach to quantify miRNA expression. The Megaplex reaction provides simultaneous reverse transcription of 450 mature miRNAs, ensuring high-throughput detection. Further, the introduction of a complementary DNA pre-amplification step significantly reduces the amount of input RNA needed, even down to single-cell level. To evaluate possible pre-amplification bias, we compared the expression of 384 miRNAs in three different cancer cell lines with Megaplex RT, with or without an additional pre-amplification step. The normalized Cq values of all three sample pairs showed a good correlation with maintenance of differential miRNA expression between the cell lines. Moreover, pre-amplification using 10 ng of input RNA enabled the detection of miRNAs that were undetectable when using Megaplex alone with 400 ng of input RNA. The high specificity of RT-qPCR together with a superior sensitivity makes this approach the method of choice for high-throughput miRNA expression profiling.
authors.affiliation: Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: Mestdagh
authors.forename: Tom
authors.initials: T
authors.lastname: Feys
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bernard
authors.forename: Simone
authors.initials: S
authors.lastname: Guenther
authors.forename: Caifu
authors.initials: C
authors.lastname: Chen
authors.forename: Frank
authors.initials: F
authors.lastname: Speleman
authors.forename: Jo
authors.initials: J
authors.lastname: Vandesompele
mesh_terms: D045744:Cell Line, Tumor; D017931:DNA Primers / Q000737:chemistry; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D006801:Humans; D035683:MicroRNAs / Q000032:analysis* / Q000378:metabolism; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins / Q000378:metabolism; D015513:Oncogene Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D017931:DNA Primers; C459801:MYCN protein, human; D035683:MicroRNAs; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins